Laminated tissue graft product

ABSTRACT

A tissue graft product comprising two or more layers of material wherein each layer comprises extracellular matrix (ECM) or polymeric material and wherein the layers are laminated together by interlocking portions of one layer with portions of another layer.

RELATED APPLICATIONS

This application is a divisional of a divisional of U.S. National Stageapplication Ser. No. 15/538,349 (allowed) which claims the benefit ofPCT Application No. PCT/NZ2015/050215, filed on Dec. 18, 2015, and U.S.Provisional Application 62/095,493 filed on Dec. 22, 2014. The entirecontents of these applications are incorporated herein by reference intheir entirety.

TECHNICAL FIELD

This invention relates to a tissue graft product useful in promoting theregrowth and healing of damaged or diseased tissue structures. Moreparticularly the invention is directed to a laminated graft productformed from multiple sheets of a biological and/or synthetic materialand to a method for making the product.

BACKGROUND OF THE INVENTION

Compositions of decellularised tissues from warm-blooded vertebrates,including humans, can be used as tissue graft materials. Common tissuegraft compositions may be derived from the dermis, the small intestine,the urinary bladder, renal capsule, the simple glandular stomach and theforestomach matrix (see, for example, U.S. Pat. Nos. 4,902,508,5,554,389, 6,099,567, 7,087,089, and 8,415,159, the entire contents ofwhich are incorporated herein by reference). These compositions areknown as extracellular matrix (ECM) and have an important role inproviding the optimal chemical and structural environment for tissuegrowth and regeneration. ECM scaffolds used for tissue regeneration aretraditionally prepared from decellularised human and animal tissuesisolated from various organs and from a variety of animal connectivetissue and basement membrane sources. These scaffolds promote tissueregeneration and are well-tolerated immunologically.

The ideal tissue graft is one that is the closest possible analogue tonative tissue. Tissue processing is required to remove cellularcomponents that otherwise may cause rejection and to ensure safety fromtransmissible diseases. Further processing may be introduced tocustomise the fabrication of the graft to meet site specificrequirements and to improve shelf life. Each successive processing stephas the potential to damage the ECM, alter the immune response andconsequently affect the tissue remodelling process. Chemical processing,drying and sterilisation techniques damage ECM and therefore affect thein vivo behaviour of grafts (1-3). Ready to use products are favoured bysurgeons. Consequently, minimally processed wet grafts are preferable.

One limitation of some ECM graft materials is that the thickness of thegraft is determined by the thickness of the tissue layer obtained fromthe source material. For example, the thickness of forestomach ECM sheetis limited by the thickness of the source tissue. Yet applications suchas hernia repair, skin graft, dural replacement, tendon repair andreconstructive surgery often require thicker grafts to provide adequatetensile strength, biomechanical performance and biological activity.

Individual sheets of ECM tissue typically have anisotropic mechanicalproperties that are directionally specified by the orientation ofcollagen fibres within the tissue. This results in directionalvariability in the physical properties of native single sheets of ECMtissue. Laminated graft constructs with alternate sheets havingdifferent fibre orientations can minimise the directional variability ofthe construct, and result in isotropic constructs which are stronger inmultiple directions.

To customise ECM grafts to meet site specific requirements it ispossible to fabricate laminated graft products that comprise multiplesheets of ECM using techniques such as compression and drying, chemicalcross-linking, suturing, or through the use of adhesives (see U.S. Pat.Nos. 5,885,619, 5,955,110, and 8,415,159).

Graft products made using these techniques have limitations. Where air,heat or lyophilisation is used to dry the graft products, the ECM isdamaged as a consequence of water loss. In the case of lyophilisation,ice crystal formation leads to structural changes of the scaffold (4).While dehydration and compression have been used as a method tofabricate laminated graft products, the ECM proteins are typicallydamaged in the process and consequently elicit an exaggeratedimmunological response. A further limitation of dehydrated andcompressed products is their tendency to delaminate after rehydration,during surgical handling, implantation and subsequently over time.

Sutures may be used to overcome the tendency of laminated graft productsto delaminate. However, sutures introduce a foreign material into thegraft and this can lead to inflammation, scarring and encapsulation, andmay not be desirable in some situations (5). For example, fabricating alaminated ECM graft product using permanent synthetic sutures, such asProlene, to secure the sheets together will result in the replacement ofthe ECM over time but the synthetic sutures will not be remodelled. Thisis not desirable in applications where the graft should be completelyreplaced by the patient's own tissue. Permanent sutures result in agreater likelihood of infection and limit the utility of this type ofproduct in open dermal repair applications. Graft products that includeabsorbable sutures have a limited shelf-life in a wet presentationbecause the sutures often break down through hydrolysis (6,7).Consequently, in applications that require a high tensile strength overa sustained period of time, absorbable sutures are not suitable.

Laminated graft products may also be comprised of ECM sheets heldtogether using an adhesive. However, introducing an adhesive can createa barrier to cell migration and can alter the typical mechanisms andkinetics of ECM remodelling.

Graft products that comprise chemically cross-linked sheets of ECM boundtogether are known to elicit a foreign body response and have limitedbiotrophic properties.

Graft products that are exclusively comprised of a synthetic polymermesh are known to provide long-term strength and rigidity. However, overtime the large amount of synthetic material can cause a foreign bodyresponse and may result in mesh erosion where the mesh can pass throughlayers of tissue. Synthetic polymer material grafts have no biologicalcomponent and therefore do not provide biological assistance with woundand tissue repair. This can lead to encapsulation of the graft andincreases the risk of adverse reactions.

It can therefore be seen from the problems and disadvantages associatedwith existing graft products that there is a need for laminated graftproducts that comprise ECM and/or a natural or synthetic polymermaterial and can be readily tuned to meet the biophysical requirementsof a wide range of anatomical sites and do not delaminate during surgeryor after implantation. Furthermore, a graft product that remains intactin both dry and wet presentations is desirable. A laminated graftproduct that can be presented in a wet form to avoid damage to the ECMand retain inherent biotrophic properties is most desirable.

It is therefore an object of the invention to provide a tissue graftproduct comprising two or more layers of ECM or polymeric material whichovercomes, at least in part, one or more of the abovementioned problems,or to at least provide a useful alternative to existing products orprocedures.

SUMMARY OF THE INVENTION

The invention provides a tissue graft product comprising two or morelayers of extracellular matrix (ECM) or polymeric material without theuse of any synthetic material for holding the layers together, such assutures or adhesive, thereby minimising the risk of rejection,inflammation or undesirable encapsulation.

Accordingly, in a first aspect of the invention there is provided atissue graft product comprising two or more layers of material whereineach layer comprises extracellular matrix (ECM) or polymeric materialand wherein the layers are laminated together by interlocking portionsof one layer with portions of another layer.

In certain embodiments of the invention, the tissue graft productcomprises a first layer of material having multiple lugs and a secondlayer of material having multiple holes, each lug of the first layerbeing located through a hole in the second layer, the holes and the lugshaving dimensions so that the lugs engage with a surface of the secondlayer and interlock the first layer with the second layer.

The tissue graft product may have at least one layer comprising ECM, ormay have all layers of material comprising ECM. The product may compriseany suitable number of layers of ECM and/or polymeric material, forexample 2, 3, 4, 5, 6, 7, 8, 9 or 10 layers of ECM and/or polymericmaterial. The ECM may be formed from the propria-submucosa of theforestomach of a ruminant. The tissue graft product may have at leastone layer comprising polymeric material, or may have all layers ofmaterial comprising polymeric material. The polymeric material may be asynthetic material formed from polypropylene, polytetrafluoroethylene,polyglycolic acid, polylactic acid, poliglecaprone-25, or polyester.Alternatively, the polymeric material may be a natural material such asa protein, polysaccharide, glycoprotein, proteoglycan, orglycosaminoglycan. Examples may include collagen, alginate, chitosan andsilk.

Each hole of the second layer may have any suitable shape including asubstantially circular, triangular, square, rectangular, diamond, orstar shape. Circular shaped holes typically have a diameter in the rangeof 2 to 4 mm. Each layer having multiple holes may have a density ofholes of 0.5 to 15 holes per cm².

The tissue graft product of the invention may be a substantially flatsheet or may have a 3-dimensional form shaped to conform to a locationto which the product is to be grafted.

The tissue graft product may be wet or may be dried, for example bylyophilisation.

In a second aspect, the invention provides a method of preparing atissue graft product of the invention, comprising the step of laminatingtwo or more layers of material wherein each layer comprisesextracellular matrix (ECM) or a polymeric material and wherein thelayers are laminated together by interlocking portions of one layer withportions of another layer.

In some embodiments of the invention, the method comprises the steps:

-   -   (i) applying a first layer of material having multiple lugs to a        second layer of material having multiple holes,    -   (ii) pushing the lugs of the first layer through the holes in        the second layer, the holes and the lugs having dimensions so        that the lugs engage with a surface of the second layer and        interlock the first layer with the second layer.

In another aspect of the invention, there is provided a tissue graftproduct of the invention, comprising:

-   -   (i) a mould to which a sheet of ECM or polymeric material can be        overlaid;    -   (ii) a lug cutting means for cutting lugs into a sheet of ECM or        polymeric material to form a lug sheet;    -   (iii) a piercing means for creating piercings in a sheet of ECM        or polymeric material to form a pierced sheet;    -   (iv) a means for pushing the lugs of the lug sheet through the        piercings of the pierced sheet to form the tissue graft product.

In a further aspect of the invention, there is provided the use of agraft product of the invention for replacing or repairing tissue in ahuman or other animal.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a tissue graft product of theinvention.

FIGS. 2A and 2B show cross-sectional views of schematic representationsof tissue graft products of the invention.

FIG. 3 is a schematic representation of an example of tooling used toprepare a tissue graft product of the invention.

FIG. 4 shows a process for preparing a tissue graft product of theinvention.

FIG. 5 is a schematic representation of an example of tooling used toprepare a three-dimensional tissue graft product of the invention.

FIG. 6 is a histogram showing the wet handling integrity of lugged andnon-lugged ovine forestomach matrix.

FIG. 7 is histogram showing the wet handling integrity of lugged andnon-lugged ovine reticulum matrix.

FIG. 8 is histogram showing the wet handling integrity of lugged andnon-lugged ovine pericardium matrix.

DETAILED DESCRIPTION Definitions

The term “extracellular matrix” (ECM) as used herein refers to animal orhuman tissue that has been decellularised and provides a matrix forstructural integrity and a framework for carrying other materials.

The term “decellularised” as used herein refers to the removal of cellsand their related debris from a portion of a tissue or organ, forexample, from ECM.

The term “polymeric material” as used herein refers to large moleculesor macromolecules comprising many repeated subunits, and may be naturalmaterials including, but not limited to, polypeptides and proteins (e.g.collagen), polysaccharides (e.g. alginate) and other biopolymers such asglycoproteins, or may be synthetic materials including, but not limitedto, polypropylene, polytetrafluoroethylene, polyglycolic acid,polylactic acid, and polyester.

The term “interlock” or “interlocking” as used herein refers to theengagement and fitting together of two or more overlapping sheets ofmaterial.

The term “laminate” or “laminating” as used herein refers to theoverlaying of one sheet of material on another sheet of material.

The term “sheet” as used herein refers to a substantially flat flexiblesection of ECM or polymeric material.

The term “layer” as used herein refers to two or more sheets overlaid oradjacent to and interlocked with each other.

The term “lug” as used herein refers to a section of a sheet that hasbeen partially cut out so that the lug remains fixedly attached to thesheet via a connection bridge.

The term “lug sheet” as used herein refers to a sheet into whichmultiple lugs have been cut.

The term “pierced sheet” as used herein refers to a sheet into whichmultiple holes have been pierced.

The term “lugging” as used herein refers to a process of pushing lugs ofa lug sheet through holes of a pierced sheet.

The term “lugged sheet” as used herein refers to a lug sheet where thelugs of the lug sheet have been pushed through the holes of a piercedsheet.

Laminated Graft Product

The invention relates generally to the lamination of two or more sheetsof ECM or polymeric material where the sheets are held together byinterlocking portions of one sheet with portions of another sheet.Although the invention is not restricted to any particular method ofinterlocking, the invention will be described with reference to a methodwhere lugs in one or more sheets have been pushed through holes in othersheets so that the sheets are interlocked and held together to form alaminated graft product. The laminated graft products of the inventionprovide advantages over other types of laminated products, and areuseful in a variety of clinical and therapeutic applications, includingwound repair and tissue regeneration and including applications such ashernia or ligament/tendon repair where additional tensile strength isrequired over the strength of a single sheet of ECM.

Some embodiments of the invention feature sheets laminated without theuse of any additional materials, such as sutures or adhesives, or theuse of drying steps in the process for their manufacture, which couldadversely affect or render the device unsuitable for wound or tissuerepair. These laminates have a greater tensile strength than individualsheets. They are also perforated, which facilitates the drainage offluid, reducing the risk of seroma formation.

Some laminated products described in the prior art which have beenlaminated without the addition of other compositions are unsuitable forwet presentation because they tend to delaminate under extended periodsof hydration. The product of the present invention does not delaminateafter prolonged exposure to water and retains its structural integrity.

In general terms, the invention is based on a method comprisingoverlaying multiple sheets of ECM or polymeric material and interlockingportions of the sheets to form a laminated graft product.

Extracellular Matrix

ECM-derived matrices for use in the invention are collagen-basedbiodegradable matrices comprising highly conserved collagens,glycoproteins, proteoglycans and glycosaminoglycans in their naturalconfiguration and natural concentration. One extracellular collagenousmatrix for use in this invention is ECM of a warm-blooded vertebrate.ECM can be obtained from various sources, for example, gastrointestinaltissue harvested from animals raised for meat production, includingpigs, cattle and sheep or other warm blooded vertebrates. Vertebrate ECMis a plentiful by-product of commercial meat production operations andis thus a low cost tissue graft material.

The ECM tissue suitable for use in the formation of the graft productscomprises naturally associated ECM proteins, glycoproteins and otherfactors that are found naturally within the ECM depending upon thesource of the ECM. One source of ECM tissue is the forestomach tissue ofa warm-blooded vertebrate.

Forestomach tissue is a preferred source of ECM tissue for use in thisinvention. Suitable forestomach ECM typically comprises thepropria-submucosa of the forestomach of a ruminant. In particularembodiments of the invention, the propria-submucosa is from the rumen,the reticulum or the omasum of the forestomach. These tissue scaffoldstypically have a contoured luminal surface. In one embodiment, the ECMtissue scaffold may additionally contain decellularised tissue,including portions of the epithelium, basement membrane or tunicamuscularis, and combinations thereof. The tissue scaffolds may alsocomprise one or more fibrillar proteins, including but not limited tocollagen I, collagen III or elastin, and combinations thereof. Thesesheets are known to vary in thickness and in definition depending uponthe source of vertebrate species.

Propria-submucosa tissue typically has an abluminal and a luminalsurface. The luminal surface is the surface facing the lumen of theorgan source and the abluminal surface faces the smooth muscle tissuesurface. The multiple sheets of propria-submucosa can be overlapped withthe abluminal surface contacting the luminal surface, the luminalsurface contacting the luminal surface, or with the abluminal surfacecontacting the abluminal surface of an adjacent sheet of ECM. All ofthese combinations of overlapping sheets of ECM from some or differentvertebrate or organ sources will produce a laminated graft productcomprising ECM.

One method of preparing ECM for use in accordance with this invention isdescribed in U.S. Pat. No. 8,415,159. A segment of the vertebrateforestomach, preferably harvested from ovine species is subjected to atransmural osmotic flow between two sides of the tissue, such that thetissue layers within all or a portion of the tissue are separated and/ordecellularised. The transmural osmotic flow can be directed from theluminal to the abluminal side of all or a portion of the tissue, or fromthe abluminal to the luminal side of all or a portion of the tissue.This may be achieved, for example, by separating the tissue between ahypertonic solution and a hypotonic solution, such that the transmuralosmotic flow is directed from the hypotonic solution to the hypertonicsolution. The method may further involve removing all or part of atissue layer including epithelium, basement membrane, or tunicamuscularis, and combinations thereof. The hypertonic and hypotonicsolutions may include, for example, water and optionally at least onebuffer, detergent or salt. The hypertonic solution contains a higherconcentration of solute than the hypotonic solution. In a particularembodiment, the hypertonic solution comprises 4 M NaCl and the hypotonicsolution comprises 0.28% Triton X-200 and 0.1% EDTA. In anotherparticular embodiment, the hypotonic solution comprises 0.1% SDS. Instill another embodiment, the hypotonic solution comprises 0.028% TritonX-200, 0.1% EDTA, and 0.1% SDS. The ECM can be stored in a hydrated or adehydrated state. Lyophilised or air dried ECM may be rehydrated orpartially rehydrated and used in accordance with this invention withoutsignificant loss of its biotropic and mechanical properties.

Polymeric Materials

In some embodiments of the invention, sheets of polymeric material maybe included in the product as either lug sheets or pierced sheets. Forexample, additional strength or longer persistence may be incorporatedinto the product by including a fine woven permanent synthetic materialsuch as polypropylene (found in Prolene mesh and Elevate mesh),polytetrafluoroethylene (found in Gore-Tex mesh), polyglycolic acid(found in Vicryl mesh), polylactic acid (found in Paritex progrip mesh),poliglecaprone-25 (found in Ultrapro mesh), and polyester (found inMersilene mesh). Synthetic materials such as polypropylene, PTFE andpolyester are non-resorbable and will persist indefinitely, providinglong lasting strength and rigidity. Synthetic materials such aspolyglycolic acid, polylactic acid and poliglecaprone-25 are resorbablemeshes and will provide additional strength in the short-term, but willresorb in the long term. Alternatively, the polymeric material may be anatural material, or derived from a natural material, such as proteins(e.g. collagen), polysaccharides (e.g. alginate), glycoproteins or othermaterials.

In other embodiments, the product may comprise sheets of polymericmaterial only (i.e. no sheet of ECM).

General Method for Preparing Graft Products

In one embodiment of this invention, laminated graft products are formedfrom multiple overlapped or partially overlapped sheets. The dimensionsof the individual sheets used are not critical. One method of forminglaminated graft products of ECM and/or SPM comprises the steps ofpushing a suitably shaped cutter (lug cutter) through a sheet to formnumerous lugs across the sheet to form a “lug sheet”. The lug sheet maybe a wet, dried, lyophilised, rehydrating or rehydrated sheet. A luglayer may comprise one or more combinations of lug sheets, which may ormay not overlap each other. The lug layer may also comprise differentcombinations of lug sizes and in varying patterns to improve strengthacross multiple layers of laminates. Piercing a sharp or blunt needlethrough a sheet of ECM or polymeric material to form numerous smallpiercings across the sheet forms a “pierced sheet”. A pierced sheet maybe a wet, dried, lyophilised, rehydrating or rehydrated sheet. A piercedlayer may comprise one or more combinations of pierced sheets, which mayor may not overlap each other. The lug layer is at least partiallyoverlaid onto the pierced layer. At this point in the method, the layersmay be rehydrated with a liquid such as isotonic saline. The lugs of thelug layer are then pushed through the piercings of the pierced layerusing a pin, push rod, punch, blunt needle or similar device tointerlock and secure the lug layer to the pierced layer. This forms alaminated graft product of the invention.

Referring to FIG. 1 , a laminated graft product 1 is shown comprising alug sheet 2 and three pierced sheets 3. Each section 4 shows where a lughas been cut out from the lug sheet 2 and has been pushed throughpiercings in pierced sheets 3 to the underside of the graft product 1. Asingle lug 5 is depicted having been pushed through from the lug sheet2. Other lugs are not shown. FIG. 2A is a cross-sectional view of alaminated graft product. A lug sheet 2 is shown overlaid on four piercedsheets 3. Lugs 5 are shown having been pushed through piercings in thepierced sheets 3 from the lug sheet 2. Each lug 5 remains attached tothe lug sheet 2 via a connection bridge 6. It will be appreciated thatin practice each lug 5 will lie substantially flat against the undersideof the graft product 1. All sheets are therefore interlocked and heldtogether.

In some embodiments of the invention, the laminated graft productconsists essentially of ECM tissue, free of potentially compromisingsutures, adhesives and chemical pre-treatments, and has a greatermechanical strength and collagen content per cm² than the individualsheets used to form the product.

The amount of tissue overlap between adjacent sheets can be varieddepending on the intended use, desired properties, required laminationstrength, required surface area or size of the product, provided that atleast a portion of each sheet overlaps and interlocks with a portion ofanother sheet. The lugs interlock and secure pierced sheets to the lugsheets in overlapping regions to give a laminated graft product.

The term “interlocking” or “interlock” as defined above refers to theway in which one or more lug sheets secure one or more pierced sheetstogether without the need for the addition of other materials such asadhesives or sutures, or the need for treatments such as compression anddehydration. Products formed may vary in the number of layers and sheetssuperimposed at and secured at different points of the laminated graftproduct. The variable structure of the graft products can provideenhanced mechanical strength.

The strength of the interlocked sheets of the product is dependentmainly on the surface area of overlapping lugged and pierced sheets, thedensity of fixation, the strength of the lug connection bridge, therigidity and resistance to compression of the lug, the strength of holesin the pierced sheets and the tightness of fit between the lugs andpiercings. The layers of a product with smaller piercings and largerlugs will be secured with greater strength and will therefore have areduced likelihood of lug pull-out. However, piercings that are toosmall may prevent simple lug push-though (lugging) and may be prone tofailure due to tearing of the pierced hole.

In preferred embodiments of the invention, the lug sheet compriseslyophilised tissue and can be hydrated once overlaid on a pierced sheetprior to pushing the lugs through the pierced sheet. However, in otherembodiments, the lug sheet may comprise wet, dried, rehydrating orrehydrated sheet, or combinations thereof, provided that the lug sheethas sufficient properties to resist tearing, piercing or otherdeformation that would otherwise be detrimental to the process of lugcutting and lug push through, and so that lugs do not deform and slipback out through piercings.

In some embodiments of the invention, the pierced sheet comprises wettissue. However, in other embodiments, the pierced sheet may comprisedried, lyophilised, rehydrating, rehydrated sheet, or combinationsthereof, provided that the pierced sheet has sufficient properties toresist tearing or other deformation that is detrimental to piercing, lugpush through or lug holding.

Tooling for Preparing Graft Products

In a typical method for preparing a graft product of the invention, thesheets used to prepare the lug and pierced sheets of the presentinvention are placed onto a mould. In one embodiment, the sheets can bestretched in both a longitudinal and lateral direction on a mould inorder to create tension in the sheet to allow for effective lugformation, piercing and/or lug push through. The stretched sheet ispunctured by and placed over sharp or blunt pins around the perimeter ofthe mould to maintain tension in the stretched sheet. Alternatively,sheets can be stretched and placed onto the mould and the tension in thesheets maintained using clamps or a press, or other suitable methods.

Guides such as rods, bars or similar shaped devices can be passedthrough the stretched sheet into the mould to secure the stretchedtissue to the mould and to provide a method for aligning any subsequentsheets that are added. These guides can also be used for aligning thetools used in the process, including the tools used to produce thepiercing and to push the lugs through. The use of a mould with guides inthis way ensures that lugs in the lug sheet and piercings in piercedsheets are aligned above and below each other, and that the lug pushthrough tool can also be aligned over and guided through the piercedholes.

In some embodiments, sheets are placed onto moulds and clamped in placeusing rods, bars or similar shaped devices which can be passed throughunstretched/tension free sheets. In some embodiments, a sheet is placedonto a mould and is fixed using methods including but not limited toclamps or a press.

The composition of the mould is not critical and can be designed in anysize or shape. In one embodiment, the mould is a 16 mm thick acetalblock. The mould contains clearance holes for lugs, needles and pins topass into the mould. The density, shape, size, orientation and format ofthe arrangement of clearance holes and the size, orientation, shape andformat of the clearance holes themselves can be tailored to the type,thickness and number of sheets and layers of material, and the requiredapplication. In one embodiment, clearance holes are arranged inhorizontally and vertically aligned rows and are 3.1 mm in diameter,spaced at centres of 3.5 mm. In another embodiment, clearance holes arearranged in offset horizontal or vertical rows to enable a reduction inthe force required for lug cutting and/or to allow a high density oflugs and fixation points.

In another embodiment, the mould is made from a group of small diameterrods with the ends of the rods forming the mould surface. In such anembodiment, the rod tips move to allow passage of the lugs, needlesand/or pins. In another embodiment, the mould is made of foam that partsto allow the passage of the lugs, needles and/or pins. In theseembodiments, it is unnecessary to align the mould with the processtooling.

In some embodiments, the mould has a flat planar surface and is used toform planar laminated graft products.

In a typical process, a sheet of ECM or polymeric material is placed onthe mould and lugs are cut with a suitably shaped cutter (lug cutter) toform a lug sheet. The lug sheet is removed and a new sheet of ECM orpolymeric material is placed on the mould. Alternatively, a new sheet isplaced on a different mould having a similar clearance hole and guidepattern. The new sheet is pierced using a needle or multiple needles toform a pierced sheet. Subsequently, one or more lug sheets are overlaidon to a layer of one or more pierced sheets and the lugs of the lugsheet are pushed through the layer of pierced sheets using a non-sharp(blunt end) pin or multiple pins. Once the lugs have been pushed throughthe holes of the pierced sheets, the lugs tend to open out therebyinterlocking the lug sheet and pierced sheets together to form thelaminated graft product. Graft products may be prepared having at leasttwo sheets, but may comprise 3, 4, 5, 6, 7, 8, 9, 10 or more sheets.

When wet sheets are used, a separation slip may be employed betweeneither of the sheets, lug sheets, pierced sheets and the toolingcomponents. This reduces surface tension enabling the tooling componentsto be more easily parted from the sheets and other tooling componentswithout exerting significant force on the sheets. The separation slip istypically made of a thin, flexible material, such aspolytetrafluoroethylene (PTFE) or stainless steel, which can be rolledor peeled away from the sheets to remove it without creating asignificant force on the sheets.

A hold down plate may be used to secure tissue prior to lug cutting,piercing and pressing of lugs through pierced sheets. In someembodiments, the lug cutters may be knives made from sharp metal tubes.In other embodiments, they may be made of any material which can besharpened into a blade. Lug cutters cut lugs into a circular shape butmay also be cut in any other shape such as, but not limited to, asquare, rectangle, triangle or an inverted triangle, diamond, star,leaving a connection bridge attached to the sheet.

In some embodiments, the lugs are as small as possible so that theprotruding free tips of the lugs are the least intrusive. But the lugsmust have sufficient width in order to achieve a hold. For example, alug sheet with a lug size of 2.8 mm across, spaced at centres of 3.5 mm,is preferred for preparing a 5-sheet product comprising forestomachpropria-submucosa ECM sheet. The connection bridge of each lug ispreferably as narrow as practicable while still surviving the lug pushthrough operation. For a 5-sheet product comprising forestomachpropria-submucosa ECM, a connection bridge of 0.7 mm wide is ideal. Asmaller width of lugs and connection bridges may be suitable forlaminating a smaller number of sheets, or for thinner sheets, or for theuse of a more rigid lugging material. A larger lug and connection bridgewidth may be more suitable for laminating a larger number of sheets,thicker sheets, more flexible materials or where the presence of largerfree lug tips is not a concern.

In some embodiments, lug sheets are created by cutting lugs usingmultiple lug cutters arranged in a flat block. The pattern of lugcutters will match clearance holes on the mould. This lug cutting blockis lined up with guides and clearance holes on the mould and pressureapplied to the block to cut lugs in the secured sheet on the mould toform a lug sheet. A biasing force to apply pressure can be generated byany number of suitable methods including hand force, applying a weight,an electronic press, or an hydraulic press. The lug cutting operationcan be applied multiple times on large sheets to create a larger lugsheet. In other embodiments, the lug cutters may be used individually,arranged in a flexible sheet, attached to a robotic arm, or lugs may becut using other cutting tools such as, but not limited to, a die orlaser.

In some embodiments, the piercing operation may be carried out using apolished, sharp needle with a long taper. For the lamination of a5-sheet product comprising forestomach propria-submucosa ECM sheet whereone sheet is a lug sheet having 2.8 mm wide lugs spaced at centres of3.5 mm between lugs, and having 0.7 mm wide connection bridges, a needlewith a diameter of 1.2 mm, spaced with 3.5 mm centres may be used topierce the sheets and create the holes. However, needles having adiameter of up to and even larger than 1.8 mm may produce adequateinterlocking in the finished product. A smaller needle diameter may besuitable for interlocking using a lug of smaller width or where thelugging material is more flexible.

In some embodiments, pierced sheets are created by puncturing a sheetwith a needle. Multiple needles may be arranged in a flat block. Thepattern of needles will match clearance holes on the mould. In a similarmanner to the application of force to cut lugs in a sheet to form a lugsheet, force is applied to the needle block to pierce small holes in thesheets.

The laminated product is formed by partially or completely overlappingand aligning one lug sheet over a layer of one or more pierced sheetsand performing the lugging operation. In some embodiments, one lug sheetis lugged on top of one or more pierced sheets. Lugs and piercings onthe lug and pierced sheets can be aligned by any suitable methodincluding, but not limited to, by eye, using guides or using a jig. Theinterlocked laminated graft product is formed by pushing the lugsthrough the piercings to secure the layers and sheets together.

In some embodiments, the lugging operation is carried out using one ormore blunted, rounded, and/or non-sharp pins (push-through pins). Forthe lamination of a 5-sheet product comprising forestomachpropria-submucosa ECM having one lug sheet with 2.8 mm wide lugs, spacedat centres of 3.5 mm, 0.7 mm wide connection bridges and piercings madewith 1.2 mm diameter needles, lugs may be pushed through using apush-through pin of 0.55 mm diameter. However, push-through pins up toand larger than 1 mm diameter will produce adequate interlocking.

In some embodiments, multiple push-through pins are arranged in a flatblock in a pattern that matches the pattern of clearance holes on themould. This pin block is lined up with guides and clearance holes on themould and a biasing force is applied to the block to push lugs of thelug sheet through one or more pierced sheets and into the clearanceholes, leaving the lugs in this position after the pins are retractedand removed from the sheet. This results in a graft product where pushedlugs anchor the one or more pierced sheets to the lug sheet. The luggingoperation can be applied multiple times on large laminates to create alarger laminated sheet.

FIG. 3 is a schematic representation of an example of tooling used toprepare a tissue graft product of the invention. A mould 11 (in dottedline) is shown overlaid with a sheet 12 of ECM or polymeric materialattached to the mould 11 using mould perimeter pins 13 to maintaintension in the sheet 12. A hold down plate 14 and separation slip 15 areshown clamped to the mould 11 with clamps 16. A piercing needle block 17(which alternatively may be a lug cutter block, a piercing needle blockor a push-through pin block) and a spacer block 18 may be guided ontothe mould 11 using guide rods 19. Needles 20 are held by the block 17protruding from its underside. When downward pressure is applied topiercing needle block 17, the needles 20 are forced downward to piercethe sheet 12. The piercing needle block 17 may be replaced with a lugcutter block having lug cutters protruding from its underside. In asimilar operation, when downward pressure is applied to the lug cutterblock, the lug cutters are forced downward to cut lugs into a sheet.Further, the piercing needle block 17 may instead be a push-through pinblock having push-through pins protruding from its underside. Whendownward pressure is applied to the push-through pin block, thepush-through pins are forced downward to push the lugs through piercingsin the sheet to give the interlocked laminated graft product of theinvention.

FIG. 4 shows a process for preparing a tissue graft product of theinvention. Steps A and B show the preparation of a lug sheet. Steps Cand D show the preparation of pierced sheets. Steps E and F show theconstruction of the laminated graft product. A lug cutter block 21 holdsmultiple lug cutters 22. The ends of lug cutters 22 have been cut at anangle so that when pressed through the sheet 23 of ECM or polymericmaterial held on a base 24 semi-circular cuts 25 are made in the sheet23. The lug cutter block 21 is raised to withdraw the lug cutters 22from the sheet 23, rotated 90° and forced downward to again cut thesheet 23. This is a repeated a second time so that the three-stepprocedure results in lugs 26 having been cut to form lug layer 27.Referring to step C, a piercing needle block 28 holding needles 29 isshown positioned above three sheets 30 of ECM or SPM. The piercingneedle block 28 is forced downward so that multiple piercings 31 aremade in the sheets 29 to form pierced layer 32. Lug layer 27 is overlaidon the pierced layer 32. A push-through pin block 33 holding multiplepush-through pins 34 is forced downward so that the push-through pins 34push the lugs 26 through the piercings 31 to interlock the pierced layer32 with the lug layer 27 and form the laminated graft product 35.

Graft Products Comprising Multiple Sheets

In some embodiments the product formed may comprise multiple lug sheets.Sheets may be created that have a lug pattern arrangement which isoffset to the lug pattern of other lug sheets. This avoids lugs ondifferent lug sheets being arranged on top of each other and lugs beingpushed through lug holes of overlapped lug sheets. These lug sheets cancontain piercings to allow lugs to be pushed through lug sheets and intothe pierced sheets. Sheets designed with lugs and piercings withdifferent patterns are termed “differential lug/pierced sheets”.

In other embodiments, a layer comprising one or more lug sheets and/ordifferential lug/pierced sheets may be placed over and under a layer ofone or more pierced sheets, effectively sandwiching a layer of one ormore pierced sheets between layers of sheets which contain lugs. Theresult is a thicker and stronger product. The sandwiched laminated graftproduct is formed by pushing lugs through piercings to secure the layersto form a sandwich. The lugs in the overlying lug layer are pushedthrough piercings from the top and lugs in sheets of the underlying luglayer are pushed through piercings from the bottom. Lugs may or may notbe pushed through all of the piercings and may or may not be pushedthrough the lug sheet or differentially lug/pierced sheet on theopposing side.

Referring to FIG. 2B, three pierced sheets 3 are shown sandwichedbetween a top lug sheet 7 and a bottom lug sheet 8. Lugs 9 from lugsheet 8 have been pushed through the piercings of the pierced sheets 3and lugs 10 from lug sheet 7 have also been pushed through the piercingsof the pierced sheets 3.

In a sandwiched product, lugs in overlying and underlying lug sheetsand/or differential lug/pierced sheets may be offset against each otherso that lugs from opposing sides do not come into contact. A sandwichedproduct in which lugs are not pushed through all layers or sheetsprovides advantages in situations where exposed lugs may cause tissueabrasion and/or friction. Therefore, a sandwiched device is ideal whenthe lug sheet and/or differential lug/pierced sheets comprise syntheticmaterials that are likely to be more rigid and have a greater potentialto be abrasive against tissue.

Pseudoisotropic Graft Products

In some embodiments, a pseudoisotropic laminate graft product isprepared from multiple sheets of ECM. The term “pseudoisotropic” as usedherein refers to a tissue graft material having similar physicalproperties along each axis of the graft material. The pseudoisotropiclaminate graft products of the invention may be prepared from individualsheets of ECM. ECM material is typically stronger in one directionrelative to other directions. This is often due to the alignment ofpolymer fibres (e.g. collagen) in the ECM. The method of preparing thepseudoisotropic laminate graft constructs comprises overlaying at leasta portion of a first sheet of lug or pierced sheet with a second sheetof lug or pierced sheet, where the second sheet is rotated so that thelongitudinal axis of the first sheet is at an angle relative to thelongitudinal axis of the second sheet. Additional sheets of lug orpierced sheets can be added in a similar manner to create apseudoisotropic laminate graft construct having the desired number oflaminate sheets.

Large Area Graft Products

Large area graft products can be prepared according to the invention.Since ECM is obtained from the tissue of certain animal organs there arelimitations on the size of sheets of tissue that can be used forgrafting operations. When large area sheets of graft tissue are required(e.g. for large burn wounds) sutures, adhesives or other types oftreatments must be used to create graft products having a sufficientsurface area. However, interlocking partially overlapping sheets of ECMaccording to the invention enables the preparation of laminated graftproducts having a surface area larger than the surface area of anyindividual sheet used to prepare the graft product.

Three-Dimensional Graft Products

In some embodiments, the graft product is essentially a flat flexibleproduct and has been prepared using a flat planar mould. In otherembodiments, the graft product may have a curved three-dimensionalshape. The ability to form three-dimensional shapes from sheets usingcurved moulds and lugging the sheets so that they retain their shapewithout having to introduce other materials or use other treatmentsovercomes limitations of existing technologies and is advantageous as itallows the creation of laminated products that conform more closely tothe natural shape of parts of the human body. Flat laminated graftproducts have a limited ability to conform to a naturally curved shape,such as a breast implant, and may be prone to forming folds or creases.These creases can become sites of seroma formation leading tocomplications or poorer cosmetic outcomes. Therefore, the ability toform a device into a three-dimensional conformed shape during theinterlocking process is advantageous.

Three-dimensional shaped interlocked laminated graft products may beformed using a mould with a curved shape. Moulds may also have steppededges or multiple flat surfaces arranged at different angles. The mouldmay have non-perpendicular clearance holes for use withnon-perpendicular lug cutters, needles and pins.

The lug and pierced sheets are preferably hydrated during themanufacturing process so that the tissue can more easily be conformed tothe preferred shape.

A mould with clearance holes perpendicular to the mould surface may beused with an individual lug cutter, lug cutters in a flexible sheet, lugcutters on a robotic arm, a laser or other technologies. In anotherembodiment, a lug cutting block may be used with a mould havingclearance holes which are arranged non-perpendicular to the surface.Alternatively, three-dimensional shapes can be formed by moulds withmultiple flat surfaces and a lug cutting block matching the individualflat surfaces. Similarly, a mould with clearance holes perpendicular tothe mould surface may be used with an individual needle, needles in aflexible sheet, a needle or needles on a robotic arm, a laser or othertechnologies. In another embodiment, a needle block may be used, with amould having clearance holes which are arranged non-perpendicular to thesurface. Alternatively, three-dimensional shapes can be formed by mouldswith multiple flat surfaces and a needle block matching the flatsurfaces. In the same manner, a mould with clearance holes perpendicularto the mould surface may be used with an individual pin, pins in aflexible sheet, a pin or pins on a robotic arm, or lugs may be pushedthrough piercings using other tools such as, but not limited to, a pushrod, punch, die or any non-sharp object. In another embodiment, a pinblock may be used, with a mould having clearance holes which arearranged non-perpendicular to the surface. Alternatively,three-dimensional shapes can be formed by moulds with multiple flatsurfaces and a pin block matching the flat surfaces.

The graft product may be removed from the mould and clamps by manualforce or excess tissue can be cut away from the clamps and the productcan be removed from the mould. Once the product is removed from themould, it may be dried, lyophilised, or completely hydrated or mayremain in the same hydration state as when undergoing the laminationprocess, all without risk of delamination. The product may be furthermanipulated to suit various medical applications. The product can besterilised using standard techniques.

The mechanical properties of the product can be tailored to the medicalapplication needs by adjusting the number of layers, sheets withinlayers, types of sheets within layers, modifying the shape, adjustingthe lugging pattern, size, density and shape, selection of ECM sheetsfrom varying animals and tissue sources, and the selection of SPMsheets.

Perforations enhance the in vivo remodelling properties of the grafts.Perforations are believed to promote contact of the ECM tissue withendogenous fluids and cells (by increasing the surface area of theimplanted graft). Perforations also serve as a conduit allowingextracellular fluid to pass through the graft. Perforations formedduring the manufacture of the graft product of the invention willalleviate the accumulation of fluids between the sheets of the graftconstructs by providing a conduit through which the fluid can flow outof the tissue.

FIG. 5 shows an example of tooling used to prepare a 3D tissue graftproduct of the invention. FIG. 5A shows a mould 36 on a stand 37. Themould 36 comprises multiple clearance holes 38 for receiving a needle orpin. Securing pins 39 are shown on the edge of the mould 36 to which asheet of ECM or SPM may be secured. FIG. 5B shows an alternativearrangement using multiple needles or pins. A mould 40 is shown on astand 41. A sheet 42 of ECM or SPM is shown stretched over the mould 40and secured by attachment to securing pins 43. A piercing needle block44 (which can be replaced by a push-through pin block) is attached to ablock guide 45.

Delivery of Bioactive Materials

Laminated graft products of the invention may be used to deliverbioactive materials to the graft site. The bioactive materials may beendogenous to ECM used in the preparation of a graft product or may bematerials that are incorporated into the ECM and/or polymeric materiallayers during or after the graft product manufacturing process.Bioactive materials delivered to the graft site in this way are known tobe beneficial for promoting cellular function including wound healingand other desirable physiological and pharmacological functions.

EXAMPLES Example 1: Basic Laminated Product Having One Lug Sheet andFour Pierced Sheets

ECM was prepared from vertebrate forestomach tissue in accordance withthe procedure described in U.S. Pat. No. 8,415,159. Sheets offorestomach ECM were formed from a segment of forestomach tissue of awarm-blooded vertebrate, said segment comprising the propria-submucosa.

A Lug Sheet was Prepared According to the Following Method:

four 5 mm diameter holes were punched into one sheet of lyophilised ECM,using cutting die and press, at each corner of 140 mm×140 mm square.This sheet was placed over a 140 mm×140 mm flat acetal plate with 5 mmdiameter guide rods positioned equidistant from each corner to match theholes cut into the lyophilised ECM sheet, the guide rods fixing thesideways position of the ECM on the plate. The plate has a 29 row by 29column grid of 3.1 mm diameter clearance holes positioned at 3.5 mmcentres at its centre, resulting in a perimeter section without holes ofaround 20 mm wide. This plate functioned as a mould. A hold down platewith guide holes for the lug cutters and matching the pattern ofclearance holes was placed on top of the lyophilised ECM sheet. Clampswere applied to secure the assembly together. A 5 mm thick spacer platewith a clearance hole size and pattern matching the guide rod holes andthe clearance holes was placed on top of the hold down plate. A lugcutting block comprising an acetal block with 2.5 mm diameter circulartube lug cutters spaced at centres of 3.5 mm, matching the pattern ofclearance holes was used, using guide rod holes in the acetal block andthe lug cutter guide holes to align the lug cutting block when pushingthe lug cutters through the ECM sheet. The assembly was placed in anarbour press and was compressed. The lug cutting block was retracted andremoved, rotated 180 degrees and replaced. The assembly was placed intoan arbour press and compressed. The lug cutting block was retracted and,together with the spacer, removed. The lug cutting block was rotated 90degrees and replaced. The so formed lug sheet, with lugs of around 2.8mm wide, was removed from the tooling.

Pierced Sheets were Prepared According to the Following Method:

Four overlapping sheets of fresh ECM were stretched over a flat acetalplate having the same dimensions, holes and guide rods as the platedescribed above. The plate also had pins protruding from its sides whichwere used to secure the stretched fresh ECM sheets to the plate. A 5 mmdiameter metal punch/guide was placed into each guide rod hole, throughthe fresh ECM, piercing the tissue and securing the tissue in place. A 1mm thick PTFE separation slip was placed over the fresh ECM. A hold downplate also possessing a grid of holes of size and pattern matching thatof the guide rod and clearance holes was placed on top of the PTFE slip.Clamps were applied to secure the assembly together. A piercing needleblock comprising an acetal block with size 11 embroidery machine needlesplaced in a grid of pattern matching the clearance holes was used. Thepiercing block also had guide holes aligning with the guide rods, usingthese to align the piercing needle block over the mould with the needlesfacing towards the ECM sheet. The assembly was placed in an arbour pressand was compressed. The piercing block, clamps hold down plate and PTFEseparation slip were removed.

The Laminated Graft Product was Assembled by the Following Method:

The lug sheet was placed on top of the pierced sheets, guiding the fourguide holes down the guide rods to align. The lug sheet was rehydratedwith an isotonic saline solution. The separation slip, hold down plateand clamps were replaced. A pin block comprising an acetal block withblunted pins, arranged in a grid matching that of the clearance holeswas used. The pin block also had guide holes aligning with the guiderods and was lowered on top of the hold down plate, with the pins facingtowards the ECM sheet, using the guide holes and guide rods to align.The assembly was placed in an arbour press and was compressed. The pinblock was removed, replaced and compressed in an arbour press a furthertwo times. The assembly was dissembled. A scalpel was used to cut thedevice away from the mould. This produced a 100 mm×100 mm interlockedlaminate graft construct which was moist and flexible with theappearance of a thick rough sheet. The sheet was subsequentlylyophilised to produce a rigid multi-ply device.

Example 2: Laminated Product Having Two Lug Sheets and Three PiercedSheets

Sheets of ECM were prepared as described in Example 1. Two lug sheetsand three pierced sheet were also prepared as described in Example 1.The laminated product was assembled by the following method: Two lugsheets were placed on top of the pierced sheets, guiding the four guideholes down the guide rods to align. The lug sheet was rehydrated with anisotonic saline solution. The separation slip, hold down plate andclamps were replaced. A pin block comprising an acetal block withblunted pins, arranged in a grid matching that of the clearance holeswas used. The pin block also had guide holes aligning with the guiderods and was lowered on top of the hold down plate, with the pins facingtowards the ECM sheet, using the guide holes and guide rods to align.The assembly was placed in an arbour press and was compressed. The pinblock was removed, replaced and compressed in an arbour press a furthertwo times. The assembly was dissembled. A scalpel was used to cut thedevice away from the mould. This produced a 100 mm×100 mm laminatedproduct that was moist and flexible having the appearance of a thickrough sheet.

Example 3: Laminated Product Having a Polypropylene Lug Sheet

Sheets of ECM were prepared as described in Example 1. A lug sheet wasprepared as described in Example 1 except that the sheet used waspolypropylene mesh. Pierced sheets were prepared as described inExample 1. The laminated product was prepared by the following method:The polypropylene lug sheet was placed on top of the pierced sheets,guiding the four guide holes down the guide rods to align. The lug sheetwas rehydrated with an isotonic saline solution. The separation slip,hold down plate and clamps were replaced. A pin block comprising anacetal block with blunted pins, arranged in a grid matching that of theclearance holes was used. The pin block also had guide holes aligningwith the guide rods and was lowered on top of the hold down plate, withthe pins facing towards the ECM sheet, using the guide holes and guiderods to align. The assembly was placed in an arbour press and wascompressed. The pin block was removed, replaced and compressed in anarbour press a further two times. The assembly was dissembled. A scalpelwas used to cut the device away from the mould. This produced a 100mm×100 mm interlocked laminate graft construct which was moist and veryflexible with the appearance of a thick rough sheet.

Example 4: Laminated Product Having Different Sheets

Sheets of propria-submucosa ECM were prepared as described in Example 1.A lug sheet of lyophilised forestomach ECM was prepared as described inExample 1. Four pierced sheets were prepared according to the method ofExample 1; one composed of small intestinal propria-submucosa, onecomposed of polypropylene mesh, one composed of pericardium and onecomposed of renal capsule matrix. The laminated product was assembled bythe following method: The lug sheet was placed on top of the piercedsheets, guiding the four guide holes down the guide rods to align. Thelug sheet was rehydrated with an isotonic saline solution. Theseparation slip, hold down plate and clamps were replaced. A pin blockcomprising an acetal block with blunted pins, arranged in a gridmatching that of the clearance holes was used. The pin block also hadguide holes aligning with the guide rods and was lowered on top of thehold down plate, with the pins facing towards the ECM sheet, using theguide holes and guide rods to align. The assembly was placed in anarbour press and was compressed. The pin block was removed, replaced andcompressed in an arbour press a further two times. The assembly wasdissembled. A scalpel was used to cut the device away from the mould.This produced a 100 mm×100 mm laminated product that was moist andflexible with the appearance of a thick rough sheet. The sheet ofpolypropylene was sandwiched within the product, so as to not give theappearance of a synthetic product, and reduce the exposure of thesynthetic material at the tissue interface, while providing the benefitsof increased material strength and rigidity.

Example 5: Laminated Product Having Offset Rows of Lugs

Sheets of ECM were prepared as described in Example 1. A lug sheet oflyophilised ECM was prepared according to the method of Example 1 exceptthat the acetal plate used has the centres of clearance holes arrangedin a hexagonal lattice, each consecutive row of clearance holesstaggered to do so, providing a denser clearance hole pattern over astraight column square packing arrangement. The resulting lattice ofholes consists of 33 rows alternating between containing either 28 or 29clearance holes. The plate has a perimeter section without holes ofaround 20 mm wide. Four overlapping pierced sheets of fresh ECM wereprepared according to the method of Example 1. The laminated product wasassembled by the following method: The lug sheet was placed on top ofthe pierced sheets, guiding the four guide holes down the guide rods toalign. The lug sheet was rehydrated with an isotonic saline solution.The separation slip, hold down plate and clamps were replaced. A pinblock comprising an acetal block with blunted pins, arranged in a gridmatching that of the clearance holes was used. The pin block also hadguide holes aligning with the guide rods and was lowered on top of thehold down plate, with the pins facing towards the ECM sheet, using theguide holes and guide rods to align. The assembly was placed in anarbour press and was compressed. The pin block was removed, replaced andcompressed in an arbour press a further two times. The assembly wasdissembled. A scalpel was used to cut the device away from the mould.This produced a 100 mm×100 mm laminated product that was moist andflexible with the appearance of a thick rough sheet. Due to the offsetrow pattern of clearance holes, the force required to undertake the lugcutting, piercing and lugging operations was reduced.

Example 6: Laminated Pseudoisotropic Product

Sheets of ECM were prepared as described in Example 1. A lug sheet oflyophilised ECM was prepared according to the method of Example 1. Fouroverlapping pierced sheets of fresh ECM were prepared according to themethod of Example 1 except that they were arranged with their collagenfibre directionality at close to 36°, −72°, +72° and −36°, relative to alug sheet placed at 0°. The laminated pseudoisotropic product wasassembled by the following method: The lug sheet was placed on top ofthe pierced sheets, guiding the four guide holes down the guide rods toalign. The lug sheet was rehydrated with an isotonic saline solution.The separation slip, hold down plate and clamps were replaced. A pinblock comprising an acetal block with blunted pins, arranged in a gridmatching that of the clearance holes was used. The pin block also hadguide holes aligning with the guide rods and was lowered on top of thehold down plate, with the pins facing towards the ECM sheet, using theguide holes and guide rods to align. The assembly was placed in anarbour press and was compressed. The pin block was removed, replaced andcompressed in an arbour press a further two times. The assembly wasdissembled. A scalpel was used to cut the device away from the mould.This produced a 100 mm×100 mm laminated pseudoisotropic product that wasmoist and flexible with the appearance of a thick rough sheet. Due tothe varying fibre orientations of the individual sheets, the product hadthe appearance of a pseudoisotropic device with similar biomechanicalproperties in multiple planar directions.

Example 7: Laminated Product Having Pierced Sheets Sandwiched BetweenLug Sheets

Sheets of ECM were prepared as described in Example 1. Two lug sheets oflyophilised ECM were prepared according to the method of Example 1. Fouroverlapping pierced sheets of fresh ECM were prepared according to themethod of Example 1 except that one lug sheet was placed with the freeends of the lugs at an angle of 0° on acetal plate, before stretchingthe four overlapping sheets of fresh ECM over the mould. The result wasa pierced sheet comprising four ECM layers stretched over the mould withlugs pushed into the clearance hole cavities. The laminated sandwichproduct was assembled by the following method: The second lug sheet wasplaced on top of the pierced sheets with the free end of the lugs at anangle of 180° relative to the first lug sheet, guiding the four guideholes down the guide rods to align. The lug sheet was rehydrated with asaline solution. The separation slip, hold down plate and clamps werereplaced. A pin block comprising an acetal block with blunted pins,arranged in a grid matching that of the clearance holes but offset 0.5mm from the centres of the clearance holes and aligned so that the pinswere positioned furthest away from the free end of the lugs on thesecond lug sheet was used. The pin block also had guide holes aligningwith the guide rods and was lowered on top of the hold down plate, withthe pins facing towards the ECM sheet, using the guide holes and guiderods to align. The assembly was placed in an arbour press and wascompressed. The pin block was retracted, replaced and compressed in anarbour press a further two times. The assembly was removed from thearbour press and turned over.

A second pin block comprising an acetal block with blunted pins,arranged in a grid matching that of the clearance holes but offset 0.5mm from the centres of the clearance holes and aligned so that the pinswere positioned furthest away from the free end of the lugs on the firstlug sheet was used. The pin block also had guide holes aligning with theguide rods and was lowered on top of the mould, with the pins facingtowards the ECM sheet, using the guide holes and guide rods to align.The assembly was placed in an arbour press and was compressed. Theassembly was removed from the arbour press, the pins blocks on eitherside of the resulting construct retracted and removed, the clamps andhold down plate removed, and the separation slip removed. A scalpel wasused to cut the device away from the mould. This produced a 100 mm×100mm laminated sandwich product that was moist and very flexible with theappearance of a thick rough sheet.

Example 8: Laminated Product Having Pierced Sheets Sandwiched BetweenLug Sheets but with Lugs Hidden within Product

Sheets of ECM were prepared as described in Example 1. Two lug sheets oflyophilised ECM were prepared, one according to the method of Example 1and one according to the same method except that the mould comprised a20 row by 20 column grid of 3.1 mm diameter clearance holes positionedat 5.0 mm centres at its centre, resulting in a perimeter sectionwithout holes of around 21 mm wide. Four overlapping pierced sheets offresh ECM were prepared according to the method of Example 1 except thatone lug sheet was placed with the free ends of the lugs at an angle of0° on acetal plate, before stretching the four overlapping sheets offresh ECM over the mould. The result was a pierced sheet comprising fourECM layers stretched over the mould with lugs pushed into the clearancehole cavities. The laminated sandwich product was assembled by thefollowing method:

A pin block comprising an acetal block with a 21 row by 21 column arrayof blunted pins, arranged in a grid matching that of the clearance holeswas used. The pin block also had guide holes aligning with the guiderods and was raised from under the mould, with the pins facing towardsthe ECM sheet, using the guide holes and guide rods to align. Theassembly was placed in an arbour press and was compressed. The assemblywas removed from the arbour press, the pin block retracted and removed,the clamps and hold down plate removed, and the separation slip removed.The free ends of the lugs of the first lug sheet were flattened alongthe top of the pierced sheet in the direction they originally faced.

The second lug sheet was placed on top of the pierced sheets with thefree end of the lugs at an angle of 180° relative to the first lugsheet, guiding the four guide holes down the guide rods to align. Thelug sheet was rehydrated with a saline solution. The separation slip andhold down plate were replaced. A spacer block was placed over the holddown plate and clamps were replaced. A pin block comprising an acetalblock with a 20 row by 20 column array of blunted pins, arranged in agrid matching that of the clearance holes was used. The pin block alsohad guide holes aligning with the guide rods and was lowered on top ofthe hold down plate, with the pins facing towards the ECM sheet, usingthe guide holes and guide rods to align. The assembly was placed in anarbour press and was compressed. The pin block was retracted andcompressed in an arbour press a further two times. The assembly wasremoved from the arbour press and disassembled. The construct was cutfrom the mould using a scalpel. This produced a 100 mm×100 mminterlocked laminate graft construct which was moist and very flexiblewith the appearance of a thick rough sheet without protrusions.

Example 9: 3D Hand Laminated Product

Sheets of ECM were prepared as described in Example 1. A lug sheet wasprepared by the following method: eight 5 mm diameter holes were punchedinto one sheet of lyophilised ECM, measuring 140 mm×240 mm using acutting die and press, four down one side at centres of 10 mm from thelong edge and at centres of 10 mm, 111.5 mm, 130 mm and 231.5 mm fromone short side, and four down the only long side in mirror to the first.This sheet was placed to the left over a 140 mm×140 mm flat acetal platewith four 5 mm diameter guide rods positioned equidistant from eachcorner to match the holes cut into the left end of the lyophilised ECMsheet, the guide rods fixing the position of the ECM on the plate. Theplate has a 29 row by 29 column grid of 3.1 mm diameter clearance holespositioned at 3.5 mm centres at its centre, resulting in a perimetersection without holes of around 20 mm wide. This plate functioned as amould. A hold down plate with guide holes for the lug cutters andmatching the pattern of clearance holes was placed on top of thelyophilised ECM sheet. Clamps were applied to secure the assemblytogether. A 5 mm thick spacer plate with clearance hole size and patternmatching the guide rod holes and the clearance holes was placed on topof the hold down plate. A lug cutting block comprising an acetal blockwith 2.5 mm diameter circular tube lug cutters spaced at centres of 3.5mm, matching the pattern of clearance holes was used, using guide rodholes in the acetal block and the lug cutter guide holes to align thelug cutting block when pushing the lug cutters through the ECM sheet.The assembly was placed in an arbour press and was compressed. The lugcutting block was retracted and removed, rotated 180 degrees andreplaced. The assembly was placed into an arbour press and compressed.The lug cutting block and spacer was removed. The lug cutting block wasrotated 90 degrees and replaced. The so formed lug sheet, with lugs ofaround 2.8 mm wide covering around a 100 mm×100 mm square, was removedfrom the tooling then repositioned so that the right hand side of thesheet was over the mould. The sheet was lowered down the guide rods, thehold down plate put into position, the assembly made secure with clampsand a spacer block put into position. The lug cutter block was loweredinto position with the same orientation as used when cutting the lefthand side of the ECM sheet. The assembly was placed in an arbour pressand was compressed. The lug cutting block was retracted and removed,rotated 180 degrees and replaced. The assembly was placed into an arbourpress and compressed. The lug cutting block was retracted and, togetherwith the spacer, removed. The lug cutting block was rotated 90 degreesand replaced. The so formed lug sheet, with lugs of around 2.8 mm widecovering around a 100 mm×200 mm rectangle, was removed from the tooling.

Pierced sheets were prepared by the following method: A sheet of freshECM was stretched over a 140 mm diameter by 70 mm high semi-sphericalhollowed acetal block densely populated with 2.0 mm holes perpendicularto the semi-spherical surface, positioning the ECM sheet to take bestadvantage of any natural 3D form it possessed. The sheet of ECM was heldin the stretched position by an array of securing pins on the undersideof the block. This block had the function of a mould. Three more ECMsheets were applied, one at a time, in the same manner.

The conformed interlocked laminated graft construct was assembled by thefollowing method: The long lug sheet was partially hydrated, until itbecame flexible, and was placed on top of and stretched over the wet ECMsheets. Working along a row of lugs with two size 11 embroidery needles,each lug was lifted, a pierce was made through the four ECM layers,pushing the needle into the closest available hole in the mould, and thelug pushed through the pierce with a blunt pin. The other rows withinthe area required to be lugged were treated in the same fashion. Ascalpel was used to cut the device away from the mould. The laminatedgraft product was moist and flexible with the appearance of a thickrough sheet, and conformed to the shape of the mould.

Example 10: 3D Single Point Laminated Product

Sheets of ECM were prepared as described in Example 1. A lug sheet wasprepared by the following method: eight 5 mm diameter holes were punchedinto one sheet of lyophilised ECM, measuring 140 mm×240 mm using acutting die and press, four down one side at centres of 10 mm from thelong edge and at centres of 10 mm, 111.5 mm, 130 mm and 231.5 mm fromone short side, and four down the only long side in mirror to the first.This sheet was placed to the left over a 140 mm×140 mm flat acetal platewith four 5 mm diameter guide rods positioned equidistant from eachcorner to match the holes cut into the left end of the lyophilised ECMsheet, the guide rods fixing the position of the ECM on the plate. Theplate has a 29 row by 29 column grid of 3.1 mm diameter clearance holespositioned at 3.5 mm centres at its centre, resulting in a perimetersection without holes of around 20 mm wide. This plate functioned as amould. A hold down plate with guide holes for the lug cutters andmatching the pattern of clearance holes was placed on top of thelyophilised ECM sheet. Clamps were applied to secure the assemblytogether. A 5 mm thick spacer plate with clearance hole size and patternmatching the guide rod holes and the clearance holes was placed on topof the hold down plate. A lug cutting block comprising an acetal blockwith 2.5 mm diameter circular tube lug cutters spaced at centres of 3.5mm, matching the pattern of clearance holes was used, using guide rodholes in the acetal block and the lug cutter guide holes to align thelug cutting block when pushing the lug cutters through the ECM sheet.The assembly was placed in an arbour press and was compressed. The lugcutting block was retracted and removed, rotated 180 degrees andreplaced. The assembly was placed into an arbour press and compressed.The lug cutting block and spacer was removed. The lug cutting block wasrotated 90 degrees and replaced. The so formed lug sheet, with lugs ofaround 2.8 mm wide covering around a 100 mm×100 mm square, was removedfrom the tooling then repositioned so that the right hand side of thesheet was over the mould. The sheet was lowered down the guide rods, thehold down plate put into position, the assembly made secure with clampsand a spacer block put into position. The lug cutter block was loweredinto position with the same orientation as used when cutting the lefthand side of the ECM sheet. The assembly was placed in an arbour pressand was compressed. The lug cutting block was retracted and removed,rotated 180 degrees and replaced. The assembly was placed into an arbourpress and compressed. The lug cutting block was retracted and, togetherwith the spacer, removed. The lug cutting block was rotated 90 degreesand replaced. The so formed lug sheet, with lugs of around 2.8 mm widecovering around a 100 mm×200 mm rectangle, was removed from the tooling.

Pierced sheets were prepared by the following method: A sheet of freshECM was stretched over a 140 mm diameter by 70 high semi-sphericalhollowed acetal block populated with 2.5 mm holes perpendicular to thesemi-spherical surface and in a pattern that proved a dense array ofholes, positioning the ECM sheet to take best advantage of any natural3D form it possessed. The sheet of ECM was held in the stretchedposition by an array of securing pins on the underside of the block.This block had the function of a mould. Three more ECM sheets wereapplied, one at a time, in the same manner. A robotic arm was used topierce the sheets.

The conformed interlocked laminated graft construct was assembled by thefollowing method: The long lug sheet was partially hydrated, until itbecame flexible, and was placed on top of and stretched over the wet ECMsheets, aligning the lugs around the perimeter of the area required tobe lugged over the pierced holes. A robotic arm was used to push eachlug through the respective piercing using a blunt pin tool. A scalpelwas used to cut the device away from the mould. The laminated graftproduct was moist and flexible with the appearance of a thick roughsheet, and conformed to the shape of the mould.

Example 11: 3D Multi-Point Laminated Product

Sheets of ECM were prepared as described in Example 1. A lug sheet wasprepared by the following method: eight 5 mm diameter holes were punchedinto one sheet of lyophilised ECM, measuring 140 mm×240 mm using acutting die and press, four down one side at centres of 10 mm from thelong edge and at centres of 10 mm, 111.5 mm, 130 mm and 231.5 mm fromone short side, and four down the only long side in mirror to the first.This sheet was placed to the left over a 140 mm×140 mm flat acetal platewith four 5 mm diameter guide rods positioned equidistant from eachcorner to match the holes cut into the left end of the lyophilised ECMsheet, the guide rods fixing the position of the ECM on the plate. Theplate has a 29 row by 29 column grid of 3.1 mm diameter clearance holespositioned at 3.5 mm centres at its centre, resulting in a perimetersection without holes of around 20 mm wide. This plate functioned as amould. A hold down plate with guide holes for the lug cutters andmatching the pattern of clearance holes was placed on top of thelyophilised ECM sheet. Clamps were applied to secure the assemblytogether. A 5 mm thick spacer plate with clearance hole size and patternmatching the guide rod holes and the clearance holes was placed on topof the hold down plate. A lug cutting block comprising an acetal blockwith 2.5 mm diameter circular tube lug cutters spaced at centres of 3.5mm, matching the pattern of clearance holes was used, using guide rodholes in the acetal block and the lug cutter guide holes to align thelug cutting block when pushing the lug cutters through the ECM sheet.The assembly was placed in an arbour press and was compressed. The lugcutting block was retracted and removed, rotated 180 degrees andreplaced. The assembly was placed into an arbour press and compressed.The lug cutting block and spacer was removed. The lug cutting block wasrotated 90 degrees and replaced. The so formed lug sheet, with lugs ofaround 2.8 mm wide covering around a 100 mm×100 mm square, was removedfrom the tooling then repositioned so that the right hand side of thesheet was over the mould. The sheet was lowered down the guide rods, thehold down plate put into position, the assembly made secure with clampsand a spacer block put into position. The lug cutter block was loweredinto position with the same orientation as used when cutting the lefthand side of the ECM sheet. The assembly was placed in an arbour pressand was compressed. The lug cutting block was retracted and removed,rotated 180 degrees and replaced. The assembly was placed into an arbourpress and compressed. The lug cutting block was retracted and, togetherwith the spacer, removed. The lug cutting block was rotated 90 degreesand replaced. The so formed lug sheet, with lugs of around 2.8 mm widecovering around a 100 mm×200 mm rectangle, was removed from the tooling.

Pierced sheets were prepared by the following method: A sheet of freshECM was stretched over a 140 mm diameter by 70 high semi-sphericalhollowed acetal block populated with sections of 2.5 mm holes that wereparallel to one another and in a pattern that proved a dense array ofholes, positioning the ECM sheet to take best advantage of any natural3D form it possessed. The sheet of ECM was held in the stretchedposition by an array of securing pins on the underside of the block.This block had the function of a mould. Three more ECM sheets wereapplied, one at a time, in the same manner. A robotic arm was used topierce the sheets. The mould was indexed on a stand and a needle blockcontaining 56 size 11 embroidery needles was applied to the wet ECM, attwo positions, at each index resulting in the ECM becoming pierced withregular pattern over the area to be lugged.

The conformed interlocked laminated graft construct was assembled by thefollowing method: The long lug sheet was partially hydrated, until itbecame flexible, and was placed on top of and stretched over the wet ECMsheets, aligning the lugs around the perimeter of the area required tobe lugged over the pierced holes. The mould was indexed on a stand and apin block containing 56 blunt pins arranged in the same pattern as forthe holes in the mould was applied to the wet ECM, at two positions, ateach index resulting in the ECM becoming lugged over the area to belugged. A scalpel was used to cut the device away from the mould. Thelaminated graft product was moist and flexible with the appearance of athick rough sheet, and conformed to the shape of the mould.

Example 12: Wet Handling Integrity Test of Lugged Ovine ForestomachMatrix Laminate and Equivalent Non-Lugged Laminates

A freeze dried lugged laminate comprising one lug sheet and two piercedsheets was prepared according to Example 1, and freeze dried. Anequivalent non-lugged 3-ply laminate was prepared by lamination, inaccordance with the method of Floden et al. (8). Ovine forestomachmatrix was prepared in accordance with the procedure described in U.S.Pat. No. 8,415,159. One layer of wet ovine forestomach matrix was placedon a flat surface with the abluminal surface facing up. A second layerwas placed on top the first layer, with the smooth abluminal surfacefacing down, contacting the first layer. A third layer was placed on topof the second layer, with the smooth abluminal surface facing upward.Pressure was applied to remove any voids between the layers. Thethree-ply laminate was then lyophilised to create a three layerlaminate.

To compare the lamination strength of the lugged and equivalentnon-lugged laminates a wet handling simulation test was performed. Boththe lugged and non-lugged laminates were cut into 4 cm×4 cm samplesprior to the wet handling test. The final sample size for testing ofeach of the laminates was five (n=5). Ten petri dishes were each filledwith 20 mL of 1×PBS. At time point T=0 hours, a 4 cm×4 cm laminate(lugged or non-lugged) was introduced to a petri dish and allowed tofully rehydrate in 1×PBS prior to the wet handling test. Uponrehydration the laminate was picked up at a corner with forceps andshaken for five seconds using a side to side motion. The laminate wasthen re-introduced to the PBS and folded into quarters. While stillfolded, the laminate was then dropped from a height of 20 cm into thelid of petri dish. The laminate was then re-introduced to the PBS andshaken for a further 5 seconds. On completion of testing the extent towhich the samples delaminated was assessed. Delamination was defined as‘more than 50% of any layer became detached from the opposing layer’.Samples that delaminated following the wet handling simulation scored as‘fail’. Wet handling simulation testing was undertaken at times T=0hours, T=12 hours and 24 hours. Laminates remained submerged andhydrated in PBS for the duration of the test. The results from the wethandling simulation test are shown in FIG. 6 .

Example 13: Wet Handling Simulation Test of Ovine Reticulum LuggedLaminates and Equivalent Non-Lugged Laminates

Ovine reticulum ECM was prepared as follows. Ovine reticulum tissue wassourced from sheep, less than 1 year old. The muscle layer wasphysically separated from the tissue and discarded. The remaining tissue(approx. 1500 g) was incubated in 0.1% Triton TX-100 (10 L), withshaking at room temperature for four hours. The solution was discarded.The tissue was incubated in a solution comprising 0.1% TX-100, 0.3% TRISand 0.15% EDTA (10 L) for 18 hours at room temperature. The solution wasdiscarded. The tissue was finally rinsed in purified water (10 L) for 10min, three times at room temperature, with shaking. The decellularisedmaterial was freeze dried as required. A freeze dried lugged laminatecomprising one lug sheet and two pierced sheets was prepared accordingto Example 1, and freeze dried. An equivalent non-lugged 3-ply laminatewas prepared by according to Example 12. The wet handling simulationtest was conducted according to Example 12 to compare the laminationstrength of the lugged and equivalent non-lugged laminates. The resultsfor both lugged and non-lugged ovine reticulum ECM laminates are shownin FIG. 7 . Ovine reticulum lugged laminates did not remain intact afterinitial handling (T=0).

Example 14: Wet Handling Integrity Test of Ovine Pericardium LuggedLaminates and Equivalent Non-Lugged Laminates

Ovine pericardium ECM was prepared for lamination as follows. Ovinepericardium was sourced from sheep, less than 1 year old. Thepericardial sac was dissected from the heart and lungs and frozen toassist with fat removal from the tissue. Upon thawing, the tissue wasthen cleaned and de-ephithelialised according to Example 13. A freezedried pericardium laminate comprising one lug sheet and two piercedsheets was prepared according to Example 1 and freeze dried. Anequivalent non-lugged 3-ply laminate was prepared by according toExample 12. Notably, the pericardium geometry meant smaller devices thanthose of Examples 1 and 12. Both laminates were cut into 2 cm×2 cmlaminates prior to the wet handling test. Final sample size for eachlaminate type was four (n=4). The wet handling simulation test wasconducted according to Example 12. The results are shown in FIG. 8 .

Although the invention has been described by way of example, it shouldbe appreciated that variations and modifications may be made withoutdeparting from the scope of the invention as defined in the claims.Furthermore, where known equivalents exist to specific features, suchequivalents are incorporated as if specifically referred in thisspecification.

REFERENCES

-   1. Schenke-Layland K, Xie J, Heydarkhan-Hagvall S, Hamm-Alvarez S F,    Stock U A, Brockbank K G M, et al. Optimized Preservation of ECM in    Cardiac Tissues: Implications for Long-Term Graft Durability. Ann    Thorac Surg. 2007 May; 83(5):1641-50.-   2. Schenke-Layland K, Madershahian N, Riemann I, Starcher B,    Halbhuber K-J, König K, et al. Impact of Cryopreservation on ECM    Structures of Heart Valve Leaflets. Ann Thorac Surg. 2006 March;    81(3):918-26.-   3. Crapo P M, Gilbert T W, Badylak S F. An overview of tissue and    whole organ decellularization processes. Biomaterials. 2011 April;    32(12):3233-43.-   4. Davidenko N, Gibb T, Schuster C, Best S M, Campbell J J, Watson C    J, et al. Biomimetic collagen scaffolds with anisotropic pore    architecture. Acta Biomater. 2012 February; 8(2):667-76.-   5. Konstantinovic M L, Lagae P, Zheng F, Verbeken E K, De Ridder D,    Deprest J A. Comparison of host response to polypropylene and    non-cross-linked porcine small intestine serosal-derived collagen    implants in a rat model. BJOG Int J Obstet Gynaecol. 2005; 112(11):    1554-60.-   6. Chu C C. The effect of pH on the in vitro degradation of    poly(glycolide lactide) copolymer absorbable sutures. J Biomed Mater    Res. 1982; 16(2):117-24.-   7. Chu C C. A comparison of the effect of pH on the biodegradation    of two synthetic absorbable sutures. Ann Surg. 1982 January;    195(1):55-9.-   8. Floden E W, Malak S F, Basil-Jones M M, Negron L, Fisher J N, Lun    S, Dempsey S G, Haverkamp R G, Ward B R and May B C (2010).    Biophysical characterization of ovine forestomach extracellular    matrix biomaterials. J Biomed Mater Res B Appl Biomater 96(1):    67-75.

The invention claimed is:
 1. A method for replacing or repairing tissuein a human or other animal, comprising applying a tissue graft productto a human or other animal in need, wherein the tissue graft productcomprises two or more sheets of material wherein each sheet comprisesextracellular matrix (ECM) or polymeric material and wherein the sheetsare laminated together by interlocking portions of one sheet withportions of another sheet, wherein a first sheet has multiple lugs and asecond sheet has multiple holes, each lug of the first sheet beinglocated through a hole in the second sheet to interlock the first sheetwith the second sheet, and wherein each lug comprises a section of thefirst sheet that is cut from the first sheet and remains connected tothe first sheet.
 2. The method of claim 1, wherein the sheets are driedsheets.
 3. The method of claim 1, wherein the holes and the lugs aredimensioned so that the lugs engage with a surface of the second sheet.4. The method of claim 1, further comprising a third sheet positionedbetween the first sheet and the second sheet, wherein the third sheetcomprises multiple holes aligned with holes of the second sheet, andwherein the lugs of the first sheet extend through the holes in thethird sheet to interlock the first sheet with the second and thirdsheets.